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1 Department of Biological Sciences, Division of Environmental and Life Sciences, Macquarie University, NSW 2109, Australia
2 CSIRO Entomology, GPO Box 1700, Canberra, ACT 2601, Australia
3 Institute of Dental Research, Westmead Millennium Institute and Westmead Center for Oral Health, Westmead, NSW 2145, Australia
Correspondence
Elizabeth M. Deane
elizabeth.deane{at}anu.edu.au
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession numbers for the DNA sequences of the species or phylotypes identified in this study are EU289036–EU289066 and EU289069–EU289139.
A supplementary table of phylotypes isolated from the urogenital and anal tracts of the tammar wallaby is available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Fig. 1). This structural feature might imply that bacteria from one tract have easy access to the other. To date, the bacteria associated with these two sites have not been studied in marsupials, although in humans it has been noted that the bacteria of the urogenital tract may vary and can be influenced by bacteria growing in the anal tract and thus occasionally can cause urogenital disease (Reid et al., 2001; Tannock, 1999).
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) with more than 30 years of fundamental research into its basic biology. Despite this, little is known about the bacterial populations of this animal, all published studies having used culture-based methods that are known to be biased in favour of a small percentage of the total microbiome (Hume, 1999; Lentle et al., 2006; Old & Deane, 1998; Yadav et al., 1972). In this study we used molecular-based methods targeting the 16S rDNA gene to document the bacteria at the openings of the urogenital and anal tracts of 42 female tammar wallabies and undertook a comprehensive comparison of the major phylotypes and their diversity. This is believed to be the first extensive report of the bacterial species within the openings of these two tracts in any marsupial. The results showed that despite the close anatomical proximity of the openings of these tracts, each opening possessed a diverse and complex bacterial population comprising mainly unidentified species.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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DNA extraction.
Samples were thawed on ice and vortexed for 2 min to achieve a homogeneous suspension. A 20 µl aliquot of each sample was taken for DNA extraction and mixed with 180 µl buffer consisting of 10 mM sodium phosphate pH 6.7 containing 400 µg lysozyme (Roche Applied Science), 200 U mutanolysin (Sigma Aldrich) and 400 µg proteinase K (Qiagen), with 20 mM diethyl pyrocarbonate (Sigma Aldrich) and incubated at 56 °C for 2 h. Bacterial cells were lysed with 1 % (w/v) SDS and 200 µg RNase (Sigma Aldrich) added prior to further incubation at 37 °C for 10 min (Smith-Vaughan et al., 2006). DNA was subsequently isolated and purified using a QIAamp DNA Mini kit (Qiagen), according to the manufacturer's protocol.
Terminal restriction fragment length polymorphism analysis.
Terminal restriction fragment length polymorphism (T-RFLP) analysis (Marsh, 1999) was performed on the 16S rDNA extracted from bacteria present in the urogenital and anal swabs collected from 42 tammar wallabies. Duplicate samples of bacterial 16S rRNA genes were amplified by PCR using AmpliTaq Gold with GeneAmp (Applied Biosystems) and the fluorescently labelled 5'-hexachlorofluorescein phosphoramidite (HEX) F27 (HEX-5'-AGAGTTTGATCMTGGCTCAG-3') and the R1492 (5'-TACGGYTACCTTGTTACGACTT-3') primers (Weisburg et al., 1991). The PCR conditions were 94 °C for 10 min followed by 35 cycles of amplification at 94 °C for 30 s, 60 °C for 30 s and 72 °C for 90 s. PCR products were digested using RsaI (Promega Life Sciences) for 1 h and then deactivated at 65 °C for 15 min prior to T-RFLP analysis being performed on an Applied Biosystems 3130x1 Genetic Analyzer (Applied Biosystems) at the Macquarie University DNA Sequencing Facility.
Amplified rDNA restriction analysis and sequencing of 16S rRNA genes.
Urogenital and anal swabs from two female wallabies carrying a 1- or 4-day-old pouch young were found to generate the highest number of dissimilar terminal fragments following T-RFLP analysis and therefore were used to generate 16S rRNA gene libraries. Bacterial 16S rRNA genes were amplified by PCR as described above for T-RFLP analysis except that the forward primer, F27a, did not contain a fluorescent HEX tag and 25 amplification cycles were used. The PCR products were cloned into a T-tailed pGEM vector (Promega Life Sciences), heat shocked into Escherichia coli DH5
(Invitrogen) and the transformed E. coli grown on Luria–Bertani agar plates prior to 96 colonies being randomly selected from the urogenital and anal tracts of both wallabies to form a clone library. The inserts within the plasmid vectors were recovered by PCR using vector-specific primers pGEM-F (5'-GGCGGTCGCGGGAATTCGATT-3') and pGEM-R (5'-GCCGCGAATTCACTAGTGATT-3') (Aislabie et al., 2006) following lysis of the cells in 96-well plates at 94 °C for 30 min. The PCR conditions were 94 °C for 10 min followed by 25 cycles of 94 °C for 45 s, 55 °C for 45 s and 72 °C for 90 s.
Amplified rDNA restriction analyses (ARDRA; Grimont & Grimont, 1986) of the PCR products made use of the restriction enzymes RsaI and HaeIII to identify unique restriction patterns within the cloned 16S rRNA genes. The restriction profiles were manually compared and all unique restriction profiles sequenced. Partial 16S rDNA gene sequences of approximately 1450 bp in length, representing a specific species or phylotype, were sequenced in both directions and compared with known sequences in GenBank (http://www.ncbi.nlm.nih.gov) using the BLAST search tool (Altschul et al., 1997). Since the majority of the phylotypes were novel (<97 % sequence similarity to any known 16S rDNA sequence), they were checked to determine whether they were chimaeras. This was done by first comparing short sections of each sequence to known sequences in the GenBank database as described by Ludwig et al. (1997), and secondly determining the position of each sequence on a phylogenetic tree (see below) with respect to all other phylotypes.
Phylogenetic analysis.
The cloned 16S rDNA sequences from the anal and urogenital tracts were aligned using the CLUSTAL W program (Thompson et al., 1994) accessed through the Australian National Genome Information Service (ANGIS; http://www.angis.org.au/) and neighbourhood joining trees (Saitou & Nei, 1987) were constructed using the Jukes–Cantor parameter in the MEGA version 4 program (Tamura et al., 2007). Bootstrap analyses of 1000 replicates were used to validate the branch points in the trees.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Supplementary Table S1).
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; Marsh, 1999; Osborn et al., 2000), in silico terminal fragments from each of the 100 phylotypes were generated and compared with the 81 different terminal fragments observed in the 84 T-RFLP population profiles. This analysis identified 77 of the 81 terminal fragments as unique phylotypes. There were eight in silico phylotypes with no corresponding terminal fragments in the T-RFLP population profiles and four terminal fragments in these profiles with no corresponding in silico phylotype (Table 1).
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), as well as different HaeIII and RsaI restriction patterns on ARDRA analysis (data not shown).
Phylogenetic and community analysis
The 100 different 16S rDNA sequences comprised five different phyla. These were the Firmicutes, Proteobacteria, Actinobacteria, Fusobacteria and Bacteroidetes; their relative distribution in the two tracts is shown in Fig. 2. As a species is generally accepted to have 
97 % homology within the 16S rRNA gene(s) (Stackebrandt & Goebel, 1994), 83 % of the clones isolated were considered to be novel species as they showed <97 % homology to any known species currently in GenBank (Supplementary Table S1).
The Firmicutes were the most diverse group; they constituted 54 % of the clones isolated from the urogenital tract and 43.9 % from the anal tract (Table 2). The Firmicutes could be further divided into two classes, the Clostridia and the Lactobacillales, with the Clostridia being the predominant class in both tracts (Table 2). Eight phylotypes from the clostridial class were common to both tracts, with clone 8817-D4-A-7F representing a sizeable proportion (8.3 %) of the clones isolated from the anal tract, but only 0.6 % of the clones from the urogenital tract (Fig. 2). This phylotype was closely related to Clostridium sp. TO-931 isolated from human faecal samples and found capable of deconjugating bile acids (Wells et al., 2003).
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).
The second major phylum was the Bacteroidetes, from the point of view of both the total number of clones isolated and the extent of diversity in both tracts (Table 2). The anal and urogenital tracts shared three common phylotypes, with clone 8817-D4-C-1B being the most predominant, representing 17.8 % of the clones isolated from the urogenital tract and 21.5 % from the anal tract (Fig. 2).
The Actinobacteria and Proteobacteria phyla were represented by a similar number of clones irrespective of the tract from which they were isolated (Table 2). Three phylotypes belonging to Actinobacteria were found in both tracts, with Arcanobacterium hippocoleae consituting 10.4 % of the clones isolated from the urogenital tract and 3.4 % from the anal tract (Fig. 2). There were also two phylotypes belonging to the Proteobacteria that were found in both tracts, with Campylobacter helveticus (corresponding to clone 8817-D4-A-1C) being isolated in high numbers only from the anal tract (Fig. 2).
In contrast to the other phyla of bacteria, the Fusobacteria were only detected in the urogenital tract of the two wallabies used as the source of bacteria for ARDRA analysis, and then only at low frequency and diversity since the phylum was represented by only one clone from each of the genera Fusobacterium (clone 8817-D4-C-10F) and Leptotrichia (clone 8837-D0-C-11B; Fig. 2; Table 1). T-RFLP analysis of the 42 tammar wallabies, however, showed that these two phylotypes could potentially be isolated from both the urogenital and the anal openings within the cloaca. Clone 8817-D4-C-10F was isolated from 23.9 % of both openings while clone 8837-D0-C-11B was isolated from 76.2 % of the urogenital and 57.1 % of the anal openings, respectively.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Lentle et al., 2006; Yadav et al., 1972). For example, only 15 different bacterial colony types were observed in the stomach of the quokka, Setonix brachyurus (Moir et al., 1956), while direct visualization of smears from the vaginal cul-de-sac of the brushtail possum, Trichosurus vulpecula, detected only rod and coccal morphological types (Mahoney et al., 2002). Furthermore, culture-based studies of the forestomach of the eastern grey kangaroo, Macropus giganteus, resulted in the identification of only 17 anaerobic bacterial species, all from the phylum Firmicutes, of which five were known species (Ouwerkerk et al., 2005). More pertinently, Lentle et al. (2006) recently found fewer than five bacterial species in the gut of the tammar wallaby pouch young. Unfortunately, the conclusions drawn from these culture-based studies are known to be biased, since such methods selectively isolate 
1 % of the actual bacterial species present (Hugenholtz et al., 1998).
Analysis of the 100 sequenced phylotypes in the current study showed that 83 were novel, based on the premise that their 16S rDNAs showed <97 % sequence identity to known species or phylotypes. Most of the phylotypes belonged to two phyla, the Firmicutes and the Bacteroidetes, irrespective of the anatomical site from where they were isolated. This is consistent with the situation in humans, pigs and mice, where members of the phyla Firmicutes and the Bacteroidetes are also the two largest populations found in the distal gut (Tom-Petersen et al., 2003; Xu et al., 2007). In the tammar wallaby, the two classes of Firmicutes, the Clostridia and the Lactobacillales, represented the largest bacterial populations within the openings of the urogenital and anal tracts, from the point of view of both diversity of phylotypes and the number of clones isolated, a finding that might explain the observations of Ouwerkerk et al. (2005), who cultured only Firmicutes from the forestomach of an eastern grey kangaroo.
Among the class Lactobacillales was a predominant phylotype represented by clone 8817-D4-C-11A (Fig. 2), the 16S-rDNA of which possessed 100 % sequence identity to a S. dysgalactiae subsp. dysgalactiae strain isolated from swine (unpublished; GenBank Accession no. AB002504). Strains of this subspecies are known to be major pathogens in bovine mastitis (Denamiel et al., 2005; Park et al., 2002) and they have also recently been reported to cause bacteraemia in puppies (Vela et al., 2006). The closely related bacterium, S. dysgalactiae subsp. equisimilis (99.8 % 16S rDNA sequence identity), is also pathogenic in animals. This bacterium accounts for 5–8 % of human streptococcal wound infections as well as causing toxic shock syndrome following misuse of tampons during menstruation (Horii et al., 2006; Sachse et al., 2002). As only certain strains of the two subspecies of S. dysgalactiae are pathogenic in placental mammals (Holt et al., 1994), and as the tammar wallabies used in this study were all considered to be in good health despite the presence and colonization of their urogenital tracts with this bacterium, this may imply that this particular wallaby strain was not virulent, though this would require confirmation.
Surprisingly, no lactobacilli were detected in the opening of either the urogenital or the anal tract. This is in direct contrast to placental mammals, where lactobacilli are readily isolated from both tracts. Moreover, lactobacilli are predominant members of the resident urogenital microflora of healthy adult human females (Reid, 2000). Our observations, however, do not preclude lactobacilli being present in the urogenital tract of the tammar wallaby, as the chance of detecting a cloned 16S rDNA with 95 % confidence using a sample size of 96 (as was the case in this study) requires a species to be present at a level 3 % of the population (Chhour et al., 2005). The observation simply means that lactobacilli are not the predominant bacteria found in the urogenital tract of the female wallaby. However, as metatherian and eutherian mammals diverged from a common ancestor approximately 130 million years ago (Marshall Graves & Westerman, 2002), it is not surprising that the bacteria found in the urogenital tract of the tammar wallaby are different from those found in humans or other placental mammals. Provided a bacterial population contains the appropriate complement of genes for survival in a particular habitat, that population will proliferate independently of the presence of a particular species (Xu et al., 2007). Thus, while the bacteria found in the urogenital tract of the tammar wallaby may be different from those of placental mammals, the various roles they play and the complement of genes they possess may be similar to those of eutherian mammals. In support of this hypothesis is the recent recognition that the greatest impact on the diversity of symbiotic bacteria within different hosts is the nature of the host itself, as up to 70 % of the bacterial diversity at the species level is unique to a given host (Ley et al., 2006).
Next to the Firmicutes, members of the Bacteroidetes were the second most abundant phylum found in the openings to the urogenital and anal tracts of the tammar wallaby. Among the porphyromonads belonging to this phylum, 79.3 % of the clones were phylogenetically related to Porphyromonas cangingivalis, originally isolated from the subgingival plaque of dogs (Collins et al., 1994). Although most known porphyromonads are regarded as oral pathogens (Dommisch et al., 2007; Holt et al., 1994), the porphyromonads found in the microbiome of the tammar wallaby could possibly play a different role as they possess 91 % sequence identity to the 16S rDNA to P. cangingivalis (Supplementary Table S1). In humans, the genus Bacteroides accounts for approximately 25 % of the bacterial population of the gastrointestinal tract (Xu et al., 2004), with Bacteroides thetaiotaomicron being regarded as a potential probiotic due to its role in regulating glycan metabolism (Xu et al., 2004).
Clone 8837-D0-C-5C represented a phylotype that was also consistently isolated from the openings of the urogenital and anal tracts (Fig. 2; Supplementary Table S1). Its 16S rDNA sequence possessed 97 % identity to Arcanobacterium hippocoleae isolated from a horse with vaginitis (Hoyles et al., 2002). To our knowledge, there are only six validated species within the genus Arcanobacterium (Ohba et al., 2007). Members of this genus have been isolated from both animal and human sources (Collins et al., 1982) and include the known pathogens Arcanobacterium pyogenes and Arcanobacterium haemolyticum, and the putative opportunistic pathogen Arcanobacterium bernardiae (Billington & Jost, 2006; Funke et al., 1995). Other species within the genus, such as Arcanobacterium phocae, are of unknown pathological significance (Hoyles et al., 2002).
In the phylum Proteobacteria, the most readily isolated phylotype was represented by clone 8837-D0-A-1C, which possesses 98 % 16S rDNA identity to Campylobacter helveticus (Fig. 2; Supplementary Table S1). This phylotype was isolated only from the anal tract of the tammar wallaby. Species within the genus Campylobacter, such as Campylobacter coli and Campylobacter jejuni, are important pathogens in humans and animals (Gorkiewicz et al., 2003). However, to the best of our knowledge, C. helveticus, which was originally isolated from cat and dog faeces (Stanley et al., 1992), is not classified as a pathogen (Mandrell et al., 2005). C. helveticus is closely related to Campylobacter upsaliensis, which, although being associated with human diseases such as abscess, gastroenteritis and bacteraemia, has an ill-defined mode of pathogenicity (Moser et al., 2001).
In conclusion, the current molecular phylogenetic study showed that the bacterial populations found in the openings of the urogenital and anal tracts of the tammar wallaby are far more diverse and complex than previously reported for other anatomical sites in marsupials using simple culturing techniques. Although most of the bacteria represent potentially novel species, the majority belong to two predominant phyla, the Firmicutes and the Bacteroidetes, in a similar manner to that documented for placental mammals.
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ACKNOWLEDGEMENTS |
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Edited by: D. M. Gordon
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 11 November 2007;
revised 6 January 2008;
accepted 2 March 2008.
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